Method for assisting in differential diagnosis and treatment of autistic syndromes

ABSTRACT

A novel relationship between pancreatico-biliary secretion and autistic syndrome is disclosed. This relationship enables a novel therapy for the treatment of the symptoms of autistic syndromes, comprising the administration of a therapeutically effective, preferably intravenous, dose of secretin to an individual with autistic syndrome. The relationship further enables a differential diagnosis for autistic syndrome, comprising an analysis of an individual&#39;s blood and/or intestinal tissue for the presence of secretin and comparison of the level of secretin to known norms.

CROSS REFERENCE TO A RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/047049, filed May 19, 1997, the contents of which areincorporated herein.

FIELD OF THE INVENTION

[0002] The present invention relates in general to differentialdiagnosis and therapeutic treatment of autistic syndromes. and inparticular to a new and useful method for diagnosing and treatingautistic syndromes by measurement ad administration to secretin.

BACKGROUND OF THE INVENTION

[0003] Autistic syndrome (or autistic) is a pervasive developmentalbehavioral disorder of very early onset that is characterized be afundamental lack of normal interest in other people. (Originaldescription, Kanner L. Autistic disturbances of affective contact.Nervous Child 1943;2:217-250.) The recent diagnostic criteria (DSM IV)for autistic disorder are shown in Table 3 below from the

[0004] American Psychiatric Associations¹.

[0005] Epidemiological studies suggested a prevalence rate of autisticbehavior of approximately 2 to 5 cases in 10,000, however, recentsurveys including the entire spectrum of the disease indicate that ratesof 15 per 10,000 are a more accurate disease prevalence^(2,3). Suchfigures indicate that this disorder affects four hundred thousandAmericans, with significant social and public health costs.

[0006] Despite the substantial body of evidence implicatingneurobiological factors in the pathogenesis, precise etiolooicmechanisms of autism have yet to be identified. In the absence of aclear etiologic, although both behavioral and medical interventions areavailable to improve learning and behavior, there is no evidence of acure for autism. nor any efficient psychopharmacological treatments forthe core symptoms.

[0007] Autism is a syndrome with multiple etiologies, as is made clearboth by the evidence of neurobiological research and by the catalog ofdisorders that are present with autistic behaviors⁴. Based on clinicalobservations, there are subgroups and subtypes of subjects withsignificantly different patterns of strengths and deficits, differentpatterns of comorbidity, levels of severity, and differentpsychological/cognitive profiles. The response to therapeutic trialsalso showed a wide variety of outcomes, which may support thepossibility that there are multiple etiologies for autism. Although weknow that genetic. infectious, metabolic, immunologic,neurophysiological. and environmental causes may lead to similarpatterns of altered development with autistic behavior, the recognitionof these clear neuropathological disorders does not help us tounderstand the basic pathogenic mechanism of autism.

[0008] There is no clear biological marker of autism to allow earlydiagnosis or screening of this disease even though it is generallybelieved that early recognition and management is crucial in theprognosis. Under these circumstances, every clinical observation isimportant and may lead us to a better understanding of this disorder.

[0009] While the specific neuropathological mechanism that producesautism is unknown, it is thought to be the result of a dysfunction ofparticular groups of neurons in the central nervous system. The primarystructures implicated in the autistic disorder are the cerebellum,cerebral cortex, and medial temporal structures. One study showed asignificant loss of Purkinje-cells, and to lesser extent, of granularcells in the cerebellar hemispheres of six autistic subjects⁵. Studiesof two patients with autism showed that the hippocampal pyramidalneurons in the CA1 and CA4 fields displayed a decrease in dendriticbranching⁶. Metabolic dysfunction of cortical areas was found throughmeasurements by Single Photon Emission Computed Tomography (SPECT)⁷. Inaddition, involvement of the medial temporal lobe has been implicated beautopsy studies demonstrating increased cell density, and small cellsize in the hippocampus, amygdala. enthorhinal cortex and septalnuclei⁸. An additional argument for the temporal lobe involvement is thecase report describing a child with a left lateral olilodendroglioma,who fulfilled the criteria of autistic behaviors⁹. This case supportsthe hypothesis that damage to mesial-temporal structures at an earlydevelopmental period may lead to the autistic syndrome. Experimentalevidence also supports this argument. A two-stage removal of theamygdala-hippocampal complex in newborn monkeys resulted in behavioralchanges (abnormalities of social interactions, absence of facial andbody expressions and stereotypical behavior), resembling autism inchildren¹⁰. It is important to note that subgroups of autistic childrendisplayed distinct patterns of brain activity in the frontal andtemporal regions. Differences were more prominent in the left than theright hemisphere¹¹. Four adult patients with autism had regionallydecreased blood flow in the right lateral temporal, and in the right.left, and midfrontal lobes compared With controls¹².

[0010] The neurobiological etiology of autism is supported by theobservation that epilepsy is a common concomitant of autism¹³. affectingapproximately one-third of adults who had childhood autism Which usuallyhad began in infancy or adolescence. In addition, different subgroups ofpatients have exhibited a variety of biochemicallnimunologicalabnormalities. For example, in 20-40% of patients, whole blood serotoninlevels are elevated¹⁴, and platelet serotonin is altered. Otherobservations include changes in the levels of dopamine-beta-hydroxylase(DBH) in plasma⁵, elevations in the levels of beta-endorphin,norepinephrine,arginine-vasopressin, and abnormally low levels ofadrenocorticotropic hormone in 70% of autistic children 16, however,there are no supporting data for the autoimmune mechanism and thetherapeutic trials with steroid treatment are disappointing so far.

[0011] Drug trials for autism have included tests of the effects ofdopamine agonists. and antagonists to dopamine, serotonin and opiates,as well as beta blockers. ACTH analogs, and oxytocin¹⁸. Most of thesetreatments were associated with some beneficial effect in small groupsof patients. The broad range of biochemical abnormalities thatstimulated this wide diversity of pharmacotherapeutic trials is a clearindication that we are still far from the understanding the mainpathological events in the brain resulting in autistic behavior.

[0012] Two recent hypotheses of autism are the oploid- and the immunetheory. The opiold theory is based on the observation that the mainfeatures of autism are similar to features of opiate addiction. Theautistic-like behavior elicited by opiate administration include:reduced socialization. affective lability, repetitive stereotypedbehavior, episodes of increased motor activity, diminished crying,insensitivity to pain, and poor clinging. Motivated by this similarity,clinical trials have been conducted by using an opioid antagonist,naltrexone, in autistic patients. In an open trial, 8 to 10 childrenwere judged to show a positive response to naltrexone¹⁹. However, morerecent double-blinded studies found that naltrexone treatment failed toproduce significant changes in social behavior³⁰.

[0013] Other researchers suppose that the opioids are derived from foodsources. The enzymatic digest of casein and gluten contains peptideswith opioid activity²¹. Fukudome and Yoshikawa isolated four opioidpeptides from the digest of wheat gluten²². One of these peptidesoccurred in different sites in the primary structure of glutenin, whichis high molecular weight protein in wheat and considered as innocentprotein in celiac disease. An additional indirect argument for thepossible role of exogenous peptides was the presence of an abnormalurinary peptide pattern in patients with autism²³. Although there is noscientific evidence that these exogenous peptides may enter thebloodstream, open clinical trials in Norway have been undertaken withthe long-term elimination of gluten and casein from the diet of patientswith autistic behavior and found only mild improvement²⁴. As can be seenby prior research studies, while administration of opioids causesautistic behavior trials with specific and very restricted diets andopioid antagonists have not resulted in evident improvement in thebehavior and health of autistic patients.

[0014] Certain immune-system abnormalities have been observed inconnection with autism, such as cell-mediated immune response to humanmyelin basic protein¹⁷ and changes in the percentage of differentsubpopulations of lymphocytes²⁵. The followers of immunopathogenesistheory are trying to use large doses of steroids The administration ofsteroids resulted in some improvement in the behavior of few patients.However, to maintain this improvement continuous administration of largedoses of steroid were necessary, accompanied by all the side effect ofchronic steroid administration. A tapering of the therapeutic dosage ofsteroid resulted in an immediate relapse.

[0015] A significant portion of patients with autistic behavior alsosuffer from mild gastrointestinal symptoms, such as abdominaldistension, constipation, or chronic loose stools. Although thesegastrointestinal problems are well known, they are not considered asimportant clinical features of the autistic syndromes, nor have theybeen treated except symptomatically. Autistic children with chronicdiarrhea are not referred routinely to a pediatric gastroenterologist.In a recent study, 43% patients had altered permeability²⁶, which is astrong argument for an intestinal dysfunction in a significant portionof autistic patients.

[0016] Secretin is a 27-amino acid peptide hormone produced by theS-dells of the small intestine that are spatially distributed from theupper crypt to the villus tip, being particularly numerous in the uppertwo-thirds of the villi²⁷. The release of secretin is increased by theproducts of protein digestion, acid bathing, fat, sodium-oleate, bileand herbal extracts²⁸S (see FIG. 1A) . Secretin increases the secretionof bicarbonate in the pancreas and biliary tract, resulting in secretionof a watery, alkaline pancreatic fluid (see FIG. 1B). The effect ofsecretin on the pancreas and bile duct is mediated primarily bysecretin-induced elevation of cyclic AMP^(29,) and does not involve theinositol phosphates signal transduction pathway (see FIG. 1C).

[0017] Secretin regulates the growth and development (enzymecomposition) of the stomach, small intestine, and pancreas , andstimulates pancreatic fluid secretion, and bile secretion³¹. Inaddition, secretin has secretory, motility and circulatory effects inthe sastrointestinal tract. Secretin stimulates immunoglobulin excretionthrough bile³². Secretin increases superior mesenteric blood flow, andits distribution within the mucosa and submucosa³³, as well as lymphflow³⁴ (see Table 1).

[0018] Thus far, the clinical uses of secretin are based on itssecretory and vascular effects. The two most important diagnosticapplications are the examination of pancreatic function. and thediagnosis of gastrinoma. There is no accepted therapeutic use. A trialto use secretin in intrahepatic cholestasis in small numbers of patientsinitially was encouraging³⁵, however, a double-blind placebo-controlledmulticentric trial found no statistically significant differences in thereduction of serum bilirubin levels between secretin and placebogroups³⁶.

[0019] The structure of porcine secretin has been known for some timeand it has been isolated from porcine intestine, and has been found tobe constituted by a peptide composed of 27 amino acid residues³⁷.Moreover, it has been found that bovine and porcine secretin areidentical but that they are markedly different from chicken secretin³⁸.Although bovine and porcine secretins behave identically with humansecretin in some respects they are not structurally identical (U.S. Pat.No. 4,806,336;Carlquist et al. 2/89). U.S. Pat. No. 4,806,336 (Carlquistet al.) discloses the chemical composition of human secretin, a methodfor administering secretin for diagnostic use in determining pancreaticor gallbladder function, and a method for stimulating pancreaticsecretion in man.

[0020] There is no published information suggesting a directrelationship between autism and secretin. However, it has been proposedthat secretin and receptors of secretin are present in the brain areasthat are thought to be involved in autism. Although exactly how secretinworks in the brain is not yet fully understood, it seems likely thatsecretin regulates neurotransmitters and influences the function of avariety of cells, especially in the “hippocampal” and “amygdala” brainareas, where seem to be impaired in autism. (See Table 2.)

[0021] Our observations described in detail herein suggest that secretinis effective in the treatment of both gastrointestinal andbehavioral/developmental problems in some children with autism. Weobserved that a group of young autistic children with chronic diarrhea,while they were undergoing tests involving an injection of secretin, hadan extraordinary increase in the production of fluid from theirpancreas. During the follow- up clinical visits these same childrenshowed impressive progress in their social. behavioral and languageskills, which appears so far to be permanent. we also found that thechildren who showed these responses to injected secretin produced onlysmall amounts of their own secretin , and when given a dose of secretinby injection, they were able to produce an elevation in the blood levelof another hormone, serotonin, which has effects on the brain.

[0022] These observations demonstrate the close relationship betweensecretin and serotonin in a group of autistic children. Our findingssuggest that there are two subgroups of autistic patients, distinguishedon the basis of gastrointestinal symptoms, their own blood secretinlevels, the secretin stimulation. In addition, we found high prevalenceof other gastrointestinal abnormalities (inflammation in the esophagus,digestive enzyme deficiencies) in children with autistic behavior whichadds further support to a relationship between the presence ofgastrointestinal dysfunctions and autism.

[0023] Thus, we have discovered that the gastrointestinal/brain hormonesecretin has a beneficial therapeutic effect on the gastrointestinal andbrain function in certain autistic children. Our findings are the firstclear evidence for an association between brain and gastrointestinaldysfunctions in autistic children.

[0024] With no prior findings of secretin having the capability toinfluence human behavior, there has been no research into the effect ofsecretin on autistic disorder. This invention is based on the unique anddramatic clinical observations of the effect of secretin administeredfor the diagnostic evaluation of Gastrointestinal function in childrenwith autistic behavior. These observations included:

[0025] (1) significant improvement in the social communication(language) and behavioral skills; and

[0026] (2) hypersecretion of pancreato-biliary fluid in children withautism and chronic loose stools.

[0027] These observations described here open an entirely new directionin the autism research and may help to understand the pathogenesis ofthis disease. In addition, it may lead to a better understanding of therole of gut peptide hormones in the brain function. The existence of thegut-brain axis has been hypothesized, however, there was no clearclinical entity associated with this axis until now. This observation isthe first clear evidence for an association between gastrointestinal andbrain dysfunctions.

SUMMARY OF THE INVENTION

[0028] It is an object of the invention to provide a definitive methodfor the diagnosis of autism.

[0029] It is another object of the invention to provide an effectivetreatment for autism which does not require large doses of steroids orother medications.

[0030] It is a further object of the invention to provide an effectivetreatment for autism which does not require frequent dosage.

[0031] A particular advantage of the invention is the effectiveness ofthe treatment with a single dose of a non-steroidal hormone, and thecontinued effectiveness with follow-up doses.

[0032] These objects and others are accomplished by the stimulation ofthe pancreatico-biliary fluid secretion by the hormone, secretin, in apatient exhibiting autism. The release may follow the exogenousadministration of an effective amount of the secretin hormone itself or,alternatively, may be subsequent to the exogenous administration of asubstance that stimulates the release and/or production of secretin.

[0033] These and other objects, features, and advantages, which will beapparent from the following discussion, are achieved. in accordance withthe invention, by providing a novel, therapeutically effective,preferably intravenous, dose of secretin, so as to alleviate thesymptoms of autism in certain individuals suffering from autisticsyndromes. Additionally, oral, intramuscular, intra-articular,intradermal, subcutaneous, inhalation, and rectal routes ofadministration are believed to be effective. Our observations indicatethat the particular administration route is not critical to theinvention.

[0034] As discussed in detail herein, the preferred means of stimulatingthe release of pancreatico-biliary secretion is by intravenousadministration of a bolus of secretin in solution. However, alternate,less-invasive, routes of secretin application from external sources,such rectal and intradermal routes, are contemplated herein. As is knownin the arc, such administration would require attachment of certainbiologically acceptable chemicals to assist in the mucosal or dermalabsorption (know as permeation enhancers) and to protect againsthydrolysis by the colonic bacterial flora or other cellular enzymes.

[0035] Alternate means of stimulating secretin release, other thanexogenous administration of secretin itself, are also contemplatedherein. Specifically, as previously discussed, certain agents whendelivered orally cause the body to release secretin. For example,studies have shown that a decrease in the pH of the duodenum below 4.5results in a significant secretin release. Administration ofhydrochloric acid has been shown not only to stimulate the release ofsecretin but also to stimulate the biosynthesis of secretin [Murphy,Gastroenterology 80:1237 (1981)]. Likewise, gastric acids can triflerthe release of secretin. Therefore, it is clear that exogenousadministration 1 5 or endogenous production of acidic agents can lead tothe release of secretin as well as the endogenous production of thehormone.

[0036] Other agents linked to secretin production and/or release includebut are not limited to I- phenylpentanol or l-phenyl-l-hydroxy-N-pentane(PHP); bile salts and acids; fats and fatty acids such as sodium oleateand oleic acid; anti-ulcer compounds such as PLAUNOTOL#, 20tetraprenylacetone (TPN), geranyl-geranyl acetone (GGA), and(Z)-2-(4-methylpiperazin-1-yl)-l-[4-(2-phenvl-ethyl)phenyl]-ethyl)phenyl]-ethanone oxime hydrochloride monohydrate(MCl-727); and herbal extracts such as licorice root. Thus, it is withinthe scope of the invention to exogenously administer a substance thatcan either stimulate the release of secretin or stimulate the endogenousproduction of the hormone.

[0037] The various features of novelty which characterize the inventionare pointed out with particularity in the claims annexed to and forminga part of this disclosure. For a better under standing of the invention,its advantages and objects, reference is made to the accompanyingdescriptive matter in which a preferred embodiment of the invention isillustrated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIGS. 1A-1C: Depicts the cascade of reactions related tosecretin. FIG. 1A depicts the intestinal phase of pancreatic secretion.FIG. 1B depicts the generally accepted mechanism of bicarbonatesecretion from the pancreatic duct cell.

[0039]FIG. 1C depicts the generally accepted mechanism of proteinsecretion and cAMP and phosphorylation of regulatory proteins.

[0040]FIG. 2: Depicts the feed-back mechanism of secretin release.

[0041] FIGS. 3A-3B: Depicts the results of an Evoked Potential ResponseTest on Case 1 (JB) after an injection with secretin.

[0042]FIG. 4: Depicts pancreatico-biliary secretion of children withautism.

[0043]FIG. 5: Depicts blood secretin concentrations in autisticchildren.

[0044]FIG. 6: Depicts changes in blood serotonin concentrations inautistic children after secretin injection.

[0045]FIG. 7: Depicts secretin release after duodenal acidification inautistic children.

DETAILED DESCRIPTION OF THE INVENTION

[0046] With the exception of our data, there is not any otherobservation which links the intestinal peptide hormone, secretin, to theautistic syndrome. This is the first observation demonstrating asignificant improvement in a neurophysiological disease afteradministration of a gut-peptide. Perhaps more importantly. thesefindings should provide the basis of new group studies that focus on thebrain-sastrointestinal axis and its role in other neurologicaldisorders. As far as the secretin is concerned. it was not considered asa clinically important neuropeptide in the brain. although severalstudies suggest that secretin may influence the function of the cells inthe brain. Secretin I 0 injection was strikinly effective in increasingcircadian rise of LH and FEB secretion in ovarlecto- mized,estrogen-primed rats⁴⁰.

[0047] There are few studies demonstrating the presence and possibleproduction of secretin in the brain. Secretin-like immunoreactivity(SLI) has been identified and characterized in the pituitary,hypothalamus, pineal and septum^(41,42). The relatively highconcentration of secretin in the hypothalamus raises the possibility ofa secretinergic pathway between the brain and the neurointermediate lobeof the pituitary⁴². The concentration in the neurointerriediate lobe isabout 45 fold higher than the concentration of SLI observed in theanterior lobes.

[0048] The existence of a possible brain-gut relation is supported bethe fact that the nucleotide sequences of the coding regions of thesecretin precursor kNAs (and thus the precursor proteins) produced inthe small intestine are identical to those in brain and hypothesis⁴³.Thus. although the role of secretin in the function of CNS has not yetbeen fully elucidated, it seems likely that secretin participates inneurotransmitter regulation, and influences the function of differentcells (Table 1).

[0049] Considerable data suggest that these effects are mediated viacAMP in hypothalamus and hippocampus³⁹. TABLE 1 Physiologic Effects OfSecretin GASTROINTESTINAL TRACT CENTRAL NERVOUS SYSTEM Secretory effectsAdenlate cyclase activation (stimulatory--pancreas, biliary tract)(cAMP) Trophic effect Tyrosine hydroxylase activity (intestine,pancreas, stomach) (increase) Circulatory effect Dopamine metabolism(selective--intestine, pancreas) Motility effect Prolactin secretion(inhibitory--stomach, intestine) (increase)

[0050] Regional distribution studies indicated that the specificreceptor binding of secretin was greatest in the cerebellum,intermediate in the cortex, thalamus, striatum, hippocampus, andhypothalamus, and lowest in the midbrain and medulla/pons⁴⁴.Interestingly, secretin binding was found in the regions of the brainwhere abnormalities were found in autism (Table 2). TABLE 2 Localizationof Secretin vs brain areas with proposed dysfunction in autism.Secretin* Precursor Activates Binds to Brain area or Binds to ⁺SLI geneadenylate VPI cells Autism receptors found found cyclase receptorsCerebellum Yes Yes Cortex Yes Yes Yes Hippocampus Yes Yes Yes Yes YesAmygdaloid Yes Hypothalamus Yes Yes Yes Medulla/pons Yes Hypophvsis ?Yes Yes Thalamus Yes Yes Yes Striatum Yes Glioblasts Yes

[0051] Specific receptors for secretin have been characterized. Northernblot analysis of human tissue mRNA revealed that the relative intensityfor-expression of a 2.1 -kilobase HSR transcript was pancreas >kidney >small intestine >lung > liver, with trace levels in brain, heart, andovary. The human secretin receptor showed a homology of 80% with the ratsecretin receptor and 37% with the human type 1 vasoactive intestinalpeptide receptor⁴⁵.

[0052] There are several possible explanations for the CNS effect ofsecretin in patients with autistic spectrum disorders:

[0053] (a) secretin receptors are present in brain areas with proposeddysfunction in autism (so far, no one has examined this possibility);

[0054] (b) secretin increases the cAMP concentration in theglioblasts⁴⁶, hypothalamus⁴⁷, paraventricular nuclei, supraopticnucleus⁴⁸, and hippocampus (alterations in the hippocampal formation arestrongly suspected in autism ⁴⁹);

[0055] (c) secretin may act by activating the VIP-1receptors, which arepredominantly found in the cerebral cortex and hippocampus⁵⁰.

[0056] (d) secretin may have the same circulatory effect in the brain asin the gut, and may increase the cerebral blood flow in the area ofbrain containing secretin receptors such as hypothalamus, hippocampusand cortex (the comparison of pre- and post secretin SPECT studies inCase 1 showed a marked improvement in the cerebral blood flow aftersecretin administration, which perhaps supports such a mechanism);

[0057] (e) the hydroxylation of tyrosine to dopa is the rate-limitingreaction in catecholamine biosynthesis (it has been previously reportedthat secretin and other members of the secretin-glucagon family ofpeptides increase dopa synthesis in superior cervical ganglia invitro⁵¹; it is possible that secretin influences brain catecholaminemetabolism through activation of tyrosine hvdroxvlase); or

[0058] (t) there is a possibility of imbalance between secretin andantagonist neuropeptide hormones in the brain (for example, an imbalancebetween secretin and somatostatin, secretin and Peptide YY, and/orsecretin and glucagon, may result in an increased or decreasedsensitivity to another substance).

[0059] Secretin injected intracerebroventricularly (ICV) significantlyincreased defecation and decreased novel-object approaches in rats, butshowed no significant effects on stereotypic behavior⁵². No autistic ratmodel exists in which it could be determined whether secretin relievesautistic symptoms in rats. There is no report of secretin influencinghuman behavior. A relationship between secretin and human behavior hasnever been shown or proven.

[0060] The examples contained herein are provided for illustrativepurposes only and are in no way intended to limit the scope of thepresent invention.

[0061] Materials and Methods

[0062] Patients

[0063] Children with autistic behavior were recruited from the PediatricGastroenterology and Behavioral and Developmental Pediatric Clinics atthe University of Maryland. In each case, the diagnosis of autism wasbased on the DSM-IV criteria set forth below (Table 3) and was confirmedby pediatric neurologists experienced in evaluating pervasivedevelopmental disorders. TABLE 3 DSM-IV Criteria for Autistic DisorderA. A total of at least six items from (1), (2), and (3), with at leasttwo from (1), and one each from (2) and (3). 1. Qualitative impairmentin social interaction, as manifested by at least two of the following:a. marked impairment in the use of multiple nonverbal behaviors, such aseye-to- eye gaze, facial expression, body postures and gestures toregulate social interaction b. failure to develop peer relationshipsappropriate to developmental level c. a lack of spontaneous seeking toshare enjoyment, interests or achievements with other people (e.g.. bylack of showing, bringing, or pointing out objects of interest) d. lackof social or emotional reciprocity 2. Qualitative impairments inCommunication, as manifested by at least one of the following: a. delayin, or total lack of, the development of spoken language (notaccompanied by an attempt to compensate through alternative modes ofcommunication such as gesture or mime) b. in individuals with adequatespeech, marked impairment in the ability to initiate or sustain aconversation with others c. stereotyped and repetitive use of languageor idiosyncratic language d. lack of varied spontaneous make-believeplay or social imitative play appropriate to developmental level. 3.Restricted, repetitive, and stereotyped patterns of behavior, interests,and activities. as manifested by at least one of the following: a.Encompassing preoccupation with one or more stereotyped and restrictedpatterns of interest that is abnormal either in intensity or focus b.apparently inflexible adherence to specific, nontunctional routines orrituals c. stereotyped and repetitive motor mannerisms (e.g., hand orfinger flapping or twisting or complex whole body movements) d.persistent preoccupation with parts of objects B. Delays or abnormalfunctioning in at least one of the following areas with onset prior toage 3 years: 1. social interaction, 2. language as used in socialcommunication, or 3. symbolic or imaginative play C. Not betteraccounted for by Rett disorder or childhood disintegrative disorder.American Psychiatric Association, Diagnostic and Statistical Manual ofMental Disorders, 4th ed. 1994.

[0064] Patients were evaluated for abnormal bowel movements, evidence ofreflux esophagitis, tissue activities of digestive enzymes, volume andcontents of secreted pancreatico-biliary fluid after secretinstimulations, blood levels of peptide hormones and neurotransmitters andpresence of intestinal bacterial or candida overgrowth. The specificprocedures were as follows.

[0065] Upper Gastrointestinal Endoscopy and Pancreatic Stimulation

[0066] Chronic, non-infectious diarrhea with unclear etiology was theindication for upper gastrointestinal endoscopy. The full uppergastrointestinal work-up included biopsies for histology, measurement ofthe digestive enzymes of the small intestine (lactase, maltase, sucrase,glucoamylase) and the pancreas (amylase, lipase, trypsin, chymotrypsin).

[0067] After fasting from midnight, upper gastrointestinal endoscopieswere carried out the next morning under general anesthesia. All gastricjuice was aspirated before passing the endoscope into the duodenum. Thepancreatico-biliary juice was collected after positioning the endoscopedistal to the ampulla of Vater. An ERCP catheter was placed into thechannel of endoscope and the fluid was collected by moving the tip intothe outcome fluid and suctioning it into a syringe The pancreas was thenstimulated with secretin 2 IU/kg BW (Ferring Laboratories, Inc, Suffern.NY, USA) given intravenously within a minute. Three additional specimenswere collected after the secretin injection within a 10-minute period.Blood samples were collected prior to and 10 minutes after secretinstimulation. In a subgroup of children, the effect of duodenalacidification was determined by measurement of blood secretin levelsbefore and 4-5 minutes after washing the second part of the duodenumwith 0.05M hydrochloric acid for 5 minutes. At the end of the procedure,biopsies were obtained of the small intestine, esophagus and stomach.

[0068] Analyses

[0069] The volume of secreted fluid was calculated as ml/min and theaspirated juice analyzed for pH, protein (mg/ml; Bio-Rad protein assay),and for enzymes (amylase, trypsin, lipase, chymotrypsin, andcarboxypeptidase A and B). These enzyme assays were modified by us andrun regularly in our certified Clinical Laboratory. An aliquot ofcollected fluid was sent for bacterial and fungal culture. Intestinalbiopsy specimens were homogenized in ice-cold distilled water and theactivities of lactase, maltase, sucrase, palatinase and glucoamylasewere measured using the Dahlquist intestinal disaccharidases assay(Dahlquist, Anal. Biochem, 22:99-107 (1968); Azad Mt, Pediatr Res,1990;28: 166-170 (1990)]. The normal values were established based onmeasurements of histological normal intestinal biopsy tissues (n=104) atthe University of Maryland. In our practice, digestive enzyme activitiesbelow the established 3d percentile values are considered abnormal.

[0070] Examination of the biopsies of esophagus for reflux esophagitisused the following histological criteria: eosinophilic or lymphocyticinfiltrate in the squamous epithelium, basal layer thickening andpapillary hypertrophy. The gastric biopsies were stained with Giemsa toidentify Helicobacter pylori infection.

[0071] Blood samples were analyzed for the levels of Gastrointestinalpeptide hormones (Secretin, CCK, VIP, Peptide Y) and neurotransmitters(Serotonin. Substance P). All the assays were performed in theInterscience Institute (Inglewood, Calif.) specialized inGastrointestinal hormone measurements

[0072] Behavioral Evaluation

[0073] Prior to the secretin administration. each child underwentdevelopmental/psychological evaluation. The post-secretin evaluation wasbased upon the notes of therapists and teachers who did not know aboutthe treatment, and parent interviews and videotape recordings of childbehaviors. In addition, most underwent a structured evaluation includingassessment of intelligence, language ability, adaptive function[Vineland Adaptive Behavior Scales]⁵³ and behavioral rating scales[Child Behavior Checklist]⁵⁴. A more specific measure directly relatedto autism, the Childhood Autism Rating Scale [CARS]⁵⁵, was also used.

[0074] The most recent 8 cases had developmental/psychologicalevaluations at the University of Maryland or at the Kennedy KriegerInstitute prior to the secretin administration.

Specific Case Studies

[0075] Case 1. J.B., a 3 and {fraction (3/12)} year old boy, was theproduct of a full-term uncomplicated pregnancy. Development of languageand social behavior proceeded normally until about 15 months of age. Atthat time he lost his expressive vocabulary and his social behaviordeteriorated. He was clinically diagnosed with autism by a pediatricneurologist at 1-1/2 years of age. At 2-½ years of age, amultidisciplinary evaluation changed the diagnosis to PDD not otherwisespecified. Brainstem evoked potential studies revealed abnormalresponses to frequency modulations in sound, which suggested theinvolvement of the temporal lobes and the thalarmocortical afferents.Single photon emission computed tomography (SPECT) scan of the brainrevealed decreased perfusion in the right hemisphere, with the mostsevere decrease in the right parietal-temporal region. Because of hischronic diarrhea and elevated antigliadin IgG antibody titer, heunderwent an upper gastrointestinal endoscopy. After the administrationof 2 IU/kg BW of secretin, the patient had an extraordinary pancreaticsecretory response (10 mi/min). Three weeks after the procedure hismother reported significant changes in his gastrointestinal symptoms andbehavior. His chronic diarrhea resolved and he became potty-trained.More dramatic changes occurred in his autistic behavior (Table 4). Amongthese were improvements in eye contact, alertness, expressive language,and fine motor skills. Eight months after the procedure he has stillretained his cognitive gains. At that time, he received a second singlesecretin infusion which resulted in a further improvement in hislanguage and cognitive functions. The latest behavioral evaluationindicates that he has changed from a low functioning autistic child to asocial, non-autistic, speech-delayed child. Repeat evoked potentialstudies indicated only a minimal delay in responses eight months aftersecretin administration (see FIGS. 3A and 3B). In addition, there was aless marked decrease in perfusion of right posterior parietal and righttemporal lobes upon follow-up SPECT imaging when compared to theprevious study. TABLE 4 Changes after Secretin Stimulation in Case #1(age 3.5 years) Before Secretin Progress after Secretin administration(within two months) Two words 100's of words - will repeat someaproximation at any word requested No sentences Short sentences, such as“I love you”, “I want juice”, “Good night mammy” No flash cards 40-50Flash cards No focus on requested tasks Will sit and watch carefully.Will perform most tasks after watching once or twice. Will sort by coloror category. Will construct more complicated puzzles. Will respondappropriately to “what's this?” Diapers only Completely potty trainedWatch videos Now gets “involved” interactively with his videos. He willimitate the hand motions, sing the songs or dance to the music.Consistent sleeping problems Has slept through almost every nightentirely Infrequent (1-2 times/week) No spinning episodes “spinning”episodes Abnormal bowel movements Normal bowel movements Excessive waterconsumption Excessive water consumption - no change Limited dietpreferences No change No apparent connections Many connections madebetween new language learned and objects. Recites made between languagenames he has learned on flash cards when he sees the same on computergame and objects or video. No response to request for Responds to allkinds of requests and spontaneously gestures No interest in drawingWants to draw constantly. Will draw complete face and name the parts ashe draws. Did not imitate commands Will imitate almost any multi-stepcommand Minimal eye concact Eye contact 75% of the time

[0076] Case 2. A.S., a 5 year old boy with autism, was referred with atwo year history of diarrhea and food intolerance. His prenatal andpostnatal history were unremarkable. His autism was diagnosed be apediatric neurologist at two years and 9 months of age. According to hisparents, he appeared normal and responsive until about 2 years of age,when he completely lost his speech and no longer responded to his name.He was placed on a high dose steroid therapy at age 4 with mildimprovement. However, the beneficial effect diminished with a relapseduring the gradual steroid discontinuation. Intravenous immunoglobulinwas initiated later, but did not result in sustained benefit. During theendoscopy he received 2 IU kg body weight of secretin. There was also asignificantly increased secretory response after secretin administration(7.5 ml/min). The cognitive skills of this boy improved over a fiveweeks period following a single dosage of secretin, and he continues toprogress (Table 5). He received a second dose of secretin 3 monthslater, which caused a further improvement in his social behavior andlanguage. TABLE 5 Changes after Secretin stimulation in case #2 (age 6yrs). Category Before Secretin Changes after Secretin Time Alertness,concentration Starting into space; Self-stimulaton: does Extremelyalert: looking therapist directly in 2 days not pay attention the eye:responds immediately to commands Correct responses to Often guessing:not paying attention; 50% Responds more quickly; pays better attention:1 week drills correct responses 75-80% correct responses Receptivelanguage Variable: understands 20-30% of Retrieves objects: follows 2-3step commands: 3 weeks commands: mostly one-step commands understandswhere he is going: understands 75% of commands Expressive languageRarely repeats words Repeats 20-30% of what he hears; starting to 4-6verbalize on his own: “give me”; “come on” weeks pointing to letters onABC's and saying them “V”, “W”, “X”, “Y”, “Z”. Sleep Sleeps 7-8 hours:often wakes up at night 10-12 hours per night uninterrupted every night1 day Fine motor Very poor fine motor coordination Putting togetherLegos: turns key in door; turns 4 weeks small knobs Gross motor Willclimb steps: rides bike with Starts to hang on monkey bars: goes downfire 4 weeks assistance pole with help: riding his bike up hill and intonew areas Activity level Hyperactive even during drills: self Veryenergetic but has very good focus during 1 week simulations duringdrills and school drills Appetite Variable: eats one meal per day; eatsonly Eating more foods: 2-3 large meals per day: 1 week 1-2 foodsincreased variety of foods: wanted to eat dairy Social interaction Avoidinteractions, except with parents Hugging therapists; better eyecontact, 1 week improved mood Bowel movements Sometimes loose, palestools At first constipated for 2-3 days. then normal 2-3 dark brownstools days Movements On toes, abnormal hand and arm A decrease in mostabnormal movements but 1 week positions; flexion of left arm: motor ticsstill has hyperactivity, especially in the hands

[0077] Case 3. D.T.. a 4 year old boy, was referred for chronic diarrheawith foul smelling stools which were positive for blood. AutisticDisorder was diagnosed at age 2-1/2. He had significant delays in speechand cognitive development with limited social skills. Because of hischronic diarrhea with foul smelling stools, he was referred to anallerologist, and multiple food allergies were diagnosed. He was placedon a significantly restricted diet, without any improvement in theconsistence of his stools. He was also not potty-trained. He underwentan upper gastrointestinal endoscopy, and his pancreato-biliary responseto secretin was excessive with an output of 8 ml/minute. Histologicalstudy revealed mild reflux esophagitis. The culture of his duodenalfluid for candida and bacteria was negative. His chronic diarrhearesolved. Most of the “claimed” antigenic foods were reintroduced intohis diet without difficulty. The post-secretin behavioral evaluationswere performed at three weeks and two months after the procedure. Hislanguage, social, and communication skills improved significantly (Table6). A second infusion of secretion 3 months later accelerated hisimprovement in his language and communication skills. Category BeforeChanges Alertness, concentration Starting into space: self-stimulation(chewed Chewing clothing, hands, and licking have stopped. hands.clothing, objects): does not pay Overall more alert, he is able to stayon tasks until attention completion. Correct responses to Oftenguessing: not paving attention: 50% Rarely guessing. Very tuned in.Perhaps 80% correct drills/commands correct responses responsesCognitive Engaged in random, aimless activities Actions are morepurposeful. Playing sequencing, (dumping toys, clicking all over thecomputer matching, and memory games on the computer. screen, turning iton and off Listening to and observing stories attentively. Receptivelanguace Variable: understood 50% of commands: Understands almosteverything. Understands two-step mostly one-step commands commands.Understands and is able to respond to the question: How many? Expressivelanguage Rarely repeats words: one word only, two Repeatine almosteverything. Still no sentences, but words, no sentences has definiteinterest in finding out the names of thing 's! He tries repeatedly tomake his messace clear. Recognizes and reads a number of letters of thealphabet. Knows and can say numbers up to 10. Social interaction Shy.Slow to warm up to people. At school Now has a friend at school. Heparticipates actively in engaged in a lot of parallel play groupactivities and plays more with his brother and sister. He helps withsimple household chores when asked. He shares food with sister. Eyecontact Limited eye contact with parents, siblings More eye contact.Seeks out attention and once engaged in activity, shows good eyecontact. Sleep Difficulty settling down at times. Would not Now goes tobed willing and happily. Takes great allow bedtime storied to be read tocompletion. interest in his bedtime stories, but settles down right awaywhen told to do so. Fine motor skills Poor. Unable to use knife,scissors, crayon. Now uses fork and spoon much more readily and mouse.Seldom using spoon or Fork. He used a accurately. Uses mouse veryprecisely. Starting to spoon for eating porridge, but resisted using ithold crayon better and use scissors better. for other foods. Activitylevel. Hyperactive even during drills: self Less hyperactive. Moresettled. More attentive and simulations during drills and school.compliant to tasks. Will bounce in place on When watching videos. forexample. he would trampoline when needs to self regulate. jump up anddown and shriek with excitement. When he watches videos, he imitates.and anticipates all actions, gestures and some words, clearly followingand enjoying the story line. Appetite Was on highly restrictive diet forover a year. Normal diet seems to satisfy him more. No longer He atewell, but was frustrated by diet. Seemed needs excessive quantities andactually leaves snacks. to require enormous quantities of food to treatsin favor of activities. He eats like a normal four satisfy his appetite.year old. Bowel movements Loose stools or diarrhea Normal bowel movementsince week three Movements Jumping in response to videos, music Now hesits down and enjoys listening to music, even hums along.

[0078] Typical pancreatic juice output in children without behavioralabnormalities is approximately 1-2 ml/minute between the ages of 1 and6, and the adult response varies between 1.6 to 5.9 ml/minute^(56,75.)The average response rate in each of the three cases reported above was10, 7.5 and 8 ml/minute, respectively. Additionally, in each case,administration of secretin produced relief from autistic symptoms, andthe second injection resulted in further improvement in their behaviorand language functions.

Group Results

[0079] The number of children on whom we have collected data and/orcompleted analy sis from 10 specific tests varies and the N for eachgroup is given in the graphs, tables or texts. For certain comparisons,the children were divided into two groups based on the presence orabsence of chronic diarrhea.

[0080] Improved stool consistency

[0081] We evaluated seven children reported to have chronic loosestools. Interestingly, all of them had an improvement in the consistencyof stools after the endoscopy and secretin injection. In five childrenthis improvement was permanent; however, two had a relapse in theirdiarrhea later.

[0082] Prevalence of refill esophagitis

[0083] We asked the parents specific questions regarding unexplainedirritability or sudden aggressive behavior in their children. Six out of15 children had these symptoms. Histological examination of 11esophageal biopsy specimens provided confirming evidence of refluxesophagitis in 5 (45.5%). In addition, one child whose parents did notreport this problem also had inflammation in the esophagus. This 45.5%,incidence of esophagitis may suggest a common upper gastrointestinalmotility problem in autistic children. The prevalence of refluxesophagitis in Western countries is estimated to be only 2%⁵⁸. Childrenwith esophagitis received at least two months treatment with prokinetic(Cisapride) and H2 blocker (Zantac) medications.

[0084] Digestive enzyme anal pancreatico-biliary fluid analyses

Rate of Pancreatico-Biliary Secretion

[0085] The time required to collect basal duodenal fluid samples (1-2ml) prior to secretin administration was 2-5 minutes, which was similarto that of non-autistic controls. The average stimulatory response forsecretin in children, 3-7 years of ace, is 1-2 ml/min based on our databy using the same endoscopic collection technique. Published adultresponses usin2 duodenal intubation and extended periods of specimencollection vary between 1.5 and 4.9 ml/min^(56.57). Six out of 7autistic patients who had chronic loose stools had pancreatico-biliarysecretion rates above 5 ml/min (FIG. 4) It was observed that allchildren, after secrtion injection, had an extensive pancreato-biliarysecretory response when compared to non-autistic patients. The highestrate of secretion was 10 ml/min in one child.

pH, Protein and Enzymes

[0086] The pH of the collected fluid varied between pH 6 and 8.8. Theprotein content of basal samples was between 0.27 mg/ml and 2.46 mg/land for samples collected inmmediately after secretin injection variedbetween 0.58 and 2.9 mg/ml. The protein content of final samples (due tothe dilution effect) was between 0.62 and 1.79 mg/ml.

[0087] All of the duodenal fluid enzvme activities were within thenormal range based on assays performed in our Laboratory on specimensfrom 215 children without pancreatic disease, i.e., cystic fibrosis.

[0088] Disaccharidases and Glucoamylase

[0089] The intestinal brush border membrane enzymes—disaccharidases andglucoamylase—were measured in 12 children. Four children each had oneabnormal enzyme activity and in 1 boy the activities of two enzymes werelow (Table 7). In summary, abnormal levels in at least one of thecarbohydrate digestive enzymes was found in {fraction (5/12)} children(41.6%). The most frequent abnormality was hypolactasia (abnormally lowlactase level). TABLE 7 Abnormal disaccharidase and glucoamylase enzymeactivities in autistic children Lactase Matase Sucrase PalatinaseGlucoamylase Below 3d 3 1 0 1 1 percentile

[0090] Culture of the Duodenal Fluid

[0091] Four out of the 15 children who underwent endoscopic procedurehad urine tests for organic acid and the results suggested that theyhave intestinal veast overgrowth. Duodenal fluid specimens from 11children were tested for both bacteria and fungi (Candida) and the testsfor all the patients were notrnal, including those with positive urineorganic acid tests.

[0092] Gastrointestinal lonrnoiie andi serotonin blood levels

Blood Concentrations Prior to and After Secretin Injection

[0093] The average basal secretin level in blood was significantly lowerin children with chronic diarrhea (p<0.024) (FIG. 5). Interestingly, thebasal serotonin level after 9-12 hours of fasting was normal in allpatients (N=16), however, ten minutes after secretin injection, childrenwith chronic diarrhea showed a sigtnificant elevation in their bloodserotonin level (FIG. 6). We repeated this test in two children with thehighest elevations and they showed the same degree of response in theirblood serotonin level after secretin administration. Elevated serotoninlevels have been reported in approximately 30% of patients withautism⁵⁹⁻⁶⁵. However, it is not clear from these papers whether theserotonin was measured after fasting or following meals. While there isno change in the blood serotonin levels in healthy adult volunteersafter meals⁶⁶, our data showing that a certain fasting autistic childrenhave increased serotonin levels after secretin injection may indicatethat the serotonin levels should be measured in the fasting state in allautistic patients. It is possible that the reported increased bloodserotonin levels were the consequence of a secretin release after meals.Our finding suggest that there may be two subgroups of patients withautistic behavior based on the serotonin response to secretin injectionand possibly to meals as well. The relevance of this findinsz to theetiology of autism and to the familiar occurrence of hyperserotonemia infamrily members needs to be addressed in the future. There was no changeat all and no subgroup differences in the blood levels of VIP, substanceP or CCK after secretin injection. Peptide Y ( measured in fourchildren) showed no abnormality in blood levels either before or afterthe secretin administration.

Blood Secretin Concentrations After Duodenal Acidification

[0094] The normal physiological process of secretin release from theintestinal S-(secretin) cells has been outlined earlier (FIGS. 1-2). Weexamined this response in 5 children during endoscopy be measuringsecretin release following the acid washing of the duodenum. Thisacidification. Which decreases pH to below 4.5, should release secretininto the blood. FIG. 7 shows the secretin levels in blood before andafter 4-5 minutes of acidification. Although the sample number did notallow a clear conclusion it appears that children with low base secretinlevel less likely release secretin than children whose base level was inthe normal range.

[0095] Based on our findings, we propose there are two subgroups ofautistic patients, distinguished on the basis of gastrointestinalsymptoms, fasting blood secretin levels, and secretory responses toeither duodenal acidification or secretin injection (Table 8). TABLE 8Subgroups of patients based on the secretin and serotonin measurementsGroup I. Group II. Main gastrointestinal symptoms Chronic loosestools/Diarrhea No diarrhea Basal secretin level Low (<12 pg/ml) Normal(>12 pg/ml) Pancreatic secretion Increased (>5 ml/min) Normal (<4ml/min) Response to duodenal acidification <2 pg/ml elevation ofsecretin >2 pg/ml elevation of secretin Serotonin level after secretininjection >50% elevation <50% elevation

[0096] Behavior Evaluations

[0097] The number and type of evaluation techniques that were employedincreased across subjects as did the degree of sophistication of theprofessional staff involved in the design and conduct of the evaluationapproaches. Specifically, data on the first seven subjects consisted ofbrief clinical observations, parental report, analysis of videotapes.and reports of evaluations by professionals who had conducted routineassessments not specifically dictated by our research protocol. Whereas,data on the second eight subjects studied consisted of the same methodsdescribed above, as well as with the addition of direct observation ofbehavior employing a standard functional analysis of structuredvideotape samples (analyzed in random order by blind raters); standardassessments including Communication and Symbolic Behavior Scales (CSBS),Pre-School Language Scales—3 (PSLS), Bayley Scales of InfantDevelopment: Second Edition, Vineland Adaptive Behavior Scales,Childhood Autism Rating Scale (CARS), and Behavior Observation System(BOS). Further, the evaluation team for the last eight subjects involvedprofessionals from the areas of neuropsychology, behavioral psychologyand developmental pediatrics from both the University of Maryland Schoolof Medicine and the Kennedy Krieger Institute/Johns Hopkins UniversitySchool of Medicine.

[0098] The findings on all fifteen subjects are truly exciting. Forexample, in terms of parental report and non-protocol dictatedevaluations, dramatic changes in cognitive, social, language, and ageappropriate skills were reported for four of the first seven subjectsstudied. For one child (JB) —a 3-year, 3-month-old boy presenting withchronic diarrhea and autistic symptoms, including no eye contact and nosocial interaction—significant changes occurred in both hisGastrointestinal symptoms and behavior within three weeks after theprocedure. Specifically, his chronic diarrhea resolved and he was ableto be potty trained. Dramatic changes occurred in his autistic behaviorin terms of a dramatic improvement in eye contact, alertness, expressivelanguage and fine motor skills. Follow-up eight months after theprocedure indicated that he had retained these improvements, especiallywith regard to cognitive skills. Most impressively, the latest formalevaluation indicates that his diagnosis has changed from a lowfunctioning autistic child to a social, non-autistic, speech delayedchild. A second case (AS)—an older boy (5-years, 9-months of age) withchronic diarrhea and autistic symptomatology first noticed at two yearsof age—demonstrated progressive improvement in cognitive skills over thefive-week period following the administration of secretin. Similarly,the third case (DT)—a 4-year, 3-month-old boy with chronic diarrhea.evidence of blood in the stool, reports of allergies to seventeen foodsubstances, autistic symptoms including severe aggression, no socialinteraction, and highly distractible attention—showed post-secretinchanges including significant improvements in language, social, andcommunication skills. The last of the four cases (UP)—a 3-year,10-month-old boy with chronic diarrhea, no speech, no eye contact, and asignificant sleep disorder—improved after the secretin injection in thathe was able to be potty trained within three weeks and showedsignificant improvement in language skills in general and expressivelanguage gains in particular.

[0099] In terms of the last eight subjects studied, in addition toparental reports comparable to the first four successes. some of thestandardized test scores for this last group of subjects indicated quiteimpressive names. One child (NB) —a 3 year, 1 month-old boy withautistic symptomatology but without gastrointestinal symptoms, showed a37%7c. increase within four months on the CSBS scale as well as anincrease in language skills equivalent to five months to one near indevelopment as measured by the PSLS are lasty an 11 point increase ontie Bayley Scale of Development. The second child (SG)—3 years, 10month-old boy, who presented with chronic diarrhea. but in terms ofautistic symptomatology was one of the highest functioning childrenstudied, showed evidence of impressive gains, including a 25% increaseover a period in the CSBS, as well as six-to-nine month increases inlanguage skills, and an increase in all five areas tested on theVineland Scale of Adaptive Behaviors. A third subject (JP)—3 year, 9month-old boy with chronic diarrhea presented with a variety of autisticsymptomatology, including poor eye contact, no social interactions,self-stimulatory behavior, and delayed echolalia; but after theadministration of secretin his markedly high activity level wasdecreased by 50% as measured by actometers and he showed an increase onthe BOS of 22%. The last case described here showed both initial, smallimprovements and later quite dramatic changes post secretinadministration; specifically, this case (BA)—3 years, 8 month-old boywith chronic loose stools presented with autistic symptoms of solitaryplay, echolalia, and poor social interaction; whereas immediately aftersecretin administration, a 22% increase in the scores on the VinelandScale of Adaptive Behavior was noted as well as a delayed reactionreported by the parents some one-to-two months later that includedimproved eye contact, increased focus on tasks, as well as improvementsin lanuae.

[0100] In summary, more than half of the children who received secretinwere found to have reductions in autistic symptoms and positive chancesin cognitive, language, and social skills that were considerably greaterthan that which would be expected from developmental maturation. Thisnovel relationship between pancreato-biliary secretion and autisticdisorder enables a novel therapy for the treatment of the symptoms ofautistic syndrome's, comprising the administration of a therapeuticallyeffective. preferably intravenous, dose of secretin to an individualsuffering from autistic spectrum disorders. The relationship furtherenables a differential diagnosis for autistic syndrome, comprising ananalysis of an individual's pancreatic response, or blood/intestinalbiopsy specimen, for the presence of secretin and comparison of thelevels to known norms.

[0101] We hereby incorporate be reference our article published inJanuary 1998 in the Journal of the Association for Academic MinorityPhysicians entitled “Improved Social and Language Skills in PatientsWith Autistic Spectrum Disorders Following Secretin Administration.” Inaddition all references cited herein are incorporated by reference intheir entirety.

[0102] 1. Diagnostic and Statistical Alanual of Mental Disorders,American Psychiatric Association. 1994; 4th edition.

[0103] 2. Suiyama T. Abe T, “The Prevalence of autism in Nagoya. Japan:a total population study”, journal of Autism & Developmental Disorders.1989; 19(i):87-96.

[0104] 3. Tanoue Y. Oda S. Asano F. Kawashima K. “Epidemiology ofinfantile autism in southern Ibaraki. Japan: differences in prevalencein birth cohorts”, Journal of Autism & Developmental Disorders, 1988;18(2): 155-66.

[0105] 4. Lotspeich L, Ciaranello RD. “The neurobiology and genetics ofinfantile autism” (Review], Internatiorial Review of iVeurobiology.1993; 35:87-129.

[0106] 5. Bauman NI. Kemper TL. “Histoanaiomic observations of the brainin early infantile autism”, Neeurology. 25 1985; 35(6):866-74.

[0107] 6. Raymond GV, Bauman MIL. Kemper TL, “Hippocampus in autism: aGolgi analysis”, Acra Neuropathologica, 1996; 91(1):117-9.

[0108] 7. tvlinshew N, “In vivo brain chemistry of autism”, Niagneticresonance spectroscopy studies in The Neurobiology of Autism. NI. Baumanand T.L. Kemper (editors), The Johns Hopkins Press. Baltimore. 1994.1994:66-85.

[0109] 8. Bauman M. Kemper T. “Neuroanatomic observations of the brainin autism” in The Neurobiology of Autism, NM. Bauman and T.L. Kemper(editors), The Johns Hopkins Press. Baltimore. 1994, 1994:119-45.

[0110] 9. Hoon AH. Jr., Reiss AL. “The mesial-temporal lobe and autism:case report and review” [Review], Developmental ,Medicine & ChildNeurology. 1992; 34(3):252-9.

[0111] 10. Bachevalier J. iNlerjanian P. “The contribution of medialtemporal-lobe structures in infantile autism: a neurobehavioral study inprimates”. in Tite Neurobiology of Aittism, NI. Bauman and T.L. Kemper(editors). The Johns Hopkins Prcss. Baltimore, 1994, 1994: 146-69.

[0112] 11 . Dawvson G, Kinier LG. Panjaiotides H. Lewy A. Castelloc P,“Subgtroups of autistic children based on social beha%ior displaydistinct patterns of brain activity”, Journal of Abnormal ChildPsychology, 1995: 23(5):569-83.

[0113] 12. George M.S., Costa DC. Kouris K. Ring, HA. Ell PJ. “Cerebralblood flow abnormalities in adults with infantile autism”. Jitriiclof,Ve.r otis & Alental Disease. 199218 0( ):4 13-7.

[0114] 13. Olsson 1. Steffenbur, S. Gillbe,2 C. “Epilepsy in autism andautisticlike conditions: a population-based study”. Archives ofNeurology. 1988: 45(6):666-S.

[0115] 14. Anderson GM. Freedman DX. Cohen DJ. et al. “Whole bloodserotonin in autistic and normal subjects”, Journal of Child Psychology& Psychiatry & Allied Disciplines. 1987: 28(6): 885-900.

[0116] 15. Garnier C, Comoy E. Barthelemy C. et al.“Dopamine-beta-hydroxylase (DBH) and homovanillic acid (HVA) in autisicchildren”. Journal of Autism & Detelopmetval Disorders, 19SS: 16(1)23-9

[0117] 16. Bouvard MP. Leboyer M. Launay JM er al. “Low-dose naltrexoneeffects on plasma chemistries and clinical symptoms in autism: adouble-blind placebo-controlled study”, Psychiatry Research 1995; 58(3):191-201.

[0118] 17. Weizman R. Szekely G.A. Wijsenbeek H. Livni E, “Abnormalimmune response to brain tissue antigen in the syndrome of autism”.American Journal of Psychiatry 1982: 139 (11): 1467-5.

[0119] 18. McDougle C. Price L. Volkmar F. “Recent advances in thepharmacotherapy of autism and related conditions”, Child and AdolesceintPsychiarric Clinics of North America. 1994; 3(1):71-89

[0120] 19. Campbell M. Overall JE. Small AM. et al, “Naltrexone inautistic children: an acute open dose rance tolerance trial”. Journal ofthe American Academy of Child & Adolescent Psychiatry. 1989;28(2):200-6.

[0121] 20. Willemsen-Swinklels S.H. Buitelaar J.K. Weijnen F.G.,vanEngeland H. “Placebo-controlled acute dosage naltrexone study inyoung autistic children”, Psychiarry Research. 1995; 58(3):203-15.

[0122] 21. Zioudrou C. Streaty RA. Klee WVA. “Opioid peptides derivedfrom food proteins: the exorphins”. Jounral of Biological Chemistry,1979: 254(7):2446-9.

[0123] 22. Fukodome A. Yoshikawa M. “Opioid peptides derived from wheatgluten: their isolation and characterization”, FEBS, 1992: 296(l):107-1I.

[0124] 23. Reichelt K.L., Hole K. Hamberger A. et al. “Biologicallyactive peptide-containing fractions in schizophrenia and childhoodautism” [Review]. Advances in Biochemical Psychopharrrmacology. 1981:28:627-43.

[0125] 24. Knivsberg A-M. Wilg K, Lind G. Nodland M. Reichelt KL.“Dietary intervention in autistic svndromes”. Brain Dysfunct. 1990: 3:315-27.

[0126] 25. Warren RP. Yonk LJ, Burger RA. et al. “Deficiency ofsuppressor-inducer (CD4+CD45RA+) T cells in autism”, ImmunologicalInvestigations. 1990; 1(3):245-51.

[0127] 26. D'Eufernia P. “Abnormal intestinal permeability in childrenwith autism”. Acta Paediatrica. 1996 S5:1076-9.

[0128] 27. Inokuchi H. Fujimoto S. Hattori T. Kawai K. “Triatedthymidine radioautographic study on the origin and renewal of secretincells in the rat duodenum”, Gastroenterology, 1985: 89(5);101420.

[0129] 28. Leiter AB. Chey WY. Kopin AS. “Secrctin” in Gutt peptides.Biochemistry and Physiology edited by JH Walsh and G.J. Dockray. RavenPress. Ltd.. New York. 1994:1147-93.

[0130] 29. Lenzen R. Alpini G. Tavoloni N. “Secretin stimulates bileductular secretory activity through the cAMP system”, American Journalof Physiology. 1992; 263(4 pt 1 ):G527-32.

[0131] 30. Pollack P.F.,Wood J.G. Solomon T. “Effect of secretin ongrowth of stomach, small intestine, and pancreas of developing rats”.Digestive Diseases & Sciences, 1990; 35(6):749-58.

[0132] 31. McGill J.M. Basavappa S. Gettys TW, Fitz J.G. “Secretinactivates Cl-channels in bile duct epithelial cells through acAMP-dependent mechanism”. America Journal of Physiology. 1994; 266(4 pt1):G73 1-6.

[0133] 32. Lebenthal E. Clark B. “Immunoglobin concentrations in theduodenal fluids of infants and children II. The Effect of pancreozyminand secretin”. American Journal of Gastroenterology, 1981; 75(6):436-9.

[0134] 33. Fara J.W. Madden KS. “Effect of secretin and cholecystokininon small intestinal blood flow distribution,” American Journal orPhysiology. 1975, 29(5).1365-70.

[0135] 34. Lawrence J.A., Bryant D. Roberts K.B. Barrowman J.A. “Eftectof secretin on intestinal lymph flow and composition in the rat”.Quarteraly Journal of Experimental Physiology. 1981; 66(3):297-305.

[0136] 35. Fukumoto Y. Okita K. Yasunaga M, et a., “A new therapeutictrial of secretin in the treatment of intrahepatic cholestasis”.Gastroenterologia Japonica. 1989; 24(3):298-307.

[0137] 36. Fukumoto Y, Okita K. Kodama T. et al. “Therapeutic effect ofsecretin in patients with jaundice; double-blind placebo-controlledmulticentric trial”, Journal of Gastroemterology. 1996; 31 (3):394-403.

[0138] 37. Mutt V. Jorpes J.E. Magnusson S. “Structure of porcinesecretin: the amino acid sequence”, Eupeopean Journal of Biochemistry.1970; 15(3):513-9.

[0139] 38Carlquist M. Jornvall H. Mlutt V, “Isolation and amino acidsequence of bovine secretin”, FEBS Letters, 198S; 127(1):71-4.

[0140] 39. Fremeau R.T. Jr., Korman LY. Moody RTW, “Secretin stimulatescyclic AIMP formation in the rat brain”. Journal of Neurochemistry 1986;46(6):1947-55.

[0141] 40. Kimura F. Mitsugi N. Arita J, Akema T, Yoshida K. “Effects ofpreoptic injections of gastrin. cholecystokinin, secretin. vasoactiveintestinal peptide and PHI on the secretion of luteinizing hormone andprolactin in ovariectomized estrogen-primed rats”, Brain Research, 1987;410(2):315-22.

[0142] 41. Charlton C.G. T.L O.D. Miller R.L., Jacobowitz D.M.,“Secretin immunoreactivity in rat and pig brain”. Peptides, 1981; 2suppl 1:45-9.

[0143] 42. Charlton CG TL OD. Miller R.L. Jacobowitz D.M. “Secretin inthe rat hypothalamo-pituitary system: localization. identification andcharacterization. “Peptides, 1982; 3(3):565-7.

[0144] 43. Itoh N, Furuya T. Ozaki K. Ohta M. Kawasaki T, “The secretinprecursor gene: structure of the coding region and expression in thebrain”, Journal of Biological Chemistry. 1991: 266(19): 12595-8.

[0145] 44. Fremeau R.T., Jr., Jensen RT.Charlton C.G. Milller R.L. TLOD. Moody T.W. “Secretin: specific binding to rat brain membranes”.Journal of Neuroscience. 1983; 3(S): 162-05.

[0146] 45. Patel D.R., Kong, Y. Sreedharan S.P. “Molecular cloning andexpression of a human secretin receptor”. Molecular Pharmacology. 1995.47(3):467-73.

[0147] 46. vanCalker D. Muller M. Hamprecht B. “Regulation by secretin,vasoactive intestinal peptide, and somatostatin of cyclic AMPaccumulation in cultured brain cells”. Proceedings of te NationalAcademy of Sciences of the United States of America, 1980;77(11):6907-11.

[0148] 47. Karelson E. Laasik J. Sillard R. “Regulation of adenylatecyclase by galainin. neuropeptide Y. secretin and vasoactive intestinalpolypeptide in rat frontal cortex, hippocampus and hypothalamus”.Neuropeptides, 1995: 28(I):2 1-8.

[0149] 45. Redgate ES. Deupree J.D. Axelrod J. “Interaction ofneuropeptides and biogenic amines on cyclic adenosine monophosphateaccumulation in hypothalamic nuclei”, Brain Research. 1986: 365 (1):61-9.

[0150] 49. DeLong G.R. “Autism, amnesia, hippocampus, and learning”,[Review] Neuoscienceive & Biobehavioral Reviews. 1992: 16(1):63-70.

[0151] 50. Usdin T.B. Bonner TI. Mezey E. “Two receptors for vasoactiveintestinal polypeptide with similar speificity and complementarydistributions”. Endocrinology. 1994; 1335(6):266-80.

[0152] 51. Schwarzchild ,M.A. Zigmond R.E. Secretin and vasoactiveintestinal pepide activate tyrosine hydroxlase in sympathetic nerveendings. Journal of Neuroscience, 1989: 9(1): 160-06.

[0153] 52. Charlton C.G., Miller R.L. Crawvley J.N. Handelmann GE, TLOD,“Secretin modulation of behavioral and physiological functions in therat”. Peptides. 1983; 4(5):739-42.

[0154] 53. Sparrow SS. Balla DA. Cicchetti E.V. Vineland AdaptiveBehavior Scales: Interview Edition. American Guidance Service. CirclePines. Minn. 1984.

[0155] 54: Achenbach T.M. Mutual for the Child Behavior Checklist,4-18and 1991 Profile, Burlington V.T. University of Vermont Department ofPsychiatry, 1981.

[0156]55. Schopler E. Reichler RJ. DeVellis RF, Dalv K, “Towardobjective classification of childhood autism: Childhood Autism RatingScale (CARS)”, Journal of Autism and Developmental Disorders,198019:91-103.

[0157] 56. Dreiling D.A. Hillander F. “Studies in pancreatic functionII: a statistical study of pancreatic secretion following secretin inpatients without pancreatic disease”, Gastroenterology 1950; 15: 620.

[0158] 57. Howat H.T. Braganza J.M. “Assessment of pancreaticdysfunction in man” in The exocrine pancreas. Howat H.T., Salres H.Eds.. W.B. Saunders. Philadelphia, 1979:129.

[0159] 58. Wienbeck, M, Barnert J. Epidemiology of reflux disease andreflux esophagitis. Scan J Gastroenterol 1989; 156(Suppl):7-1 .

[0160] 59. Cook E.H. Jr., Leventhal B.L. Heller W Metz J, Wainwriaht M.Freedman D.X. Autistic children and their first-degree relatives:relationships between serotonin and norepinephrine levels andintelligence. Journal of Neuropsychiarty & Cliniical Neurosciences1990:2(3):268-74.

[0161] 60. Cook E.H. Autism: review of neurochemical investigation.Synapse 1990;6(3):292-308.

[0162] 61. Cook E.H., Leventhal B.L. The serotonin system in autism.Current Opinion in Pediatrics 1996;8(4):348-54.

[0163] 62. Cook E.H. Jr.. Arora R.C. Anderson GM. Berry-Kravis E.M. YanS.Y. Yeoh H.C. et al. Platelet serotonin studies in hyperserotonemicrelatives of children with autistic disorder. Life Sciences1993;5(25):2005-15.

[0164] 63. Bursztejn C. Ferrari P. Dreux C. Braconnier A. Lancrenon S.Metabolism of serotonin in autism in children. Encephale1988;14(6):413-9.

[0165] 64. Singh V.K. Singh EA. Warren R.P. Hyperserotoninemia andserotonin receptor antibodies in children with autism but not mentalretardation. Biological Psychiatry 1997 -11:(6):735.

[0166] 65. Abramson R.K. Wright H.H. Carpenter R. Brennan W. Lumpuy O.Cole E. et al. Elevated blood serotonin in autistic probands and theirfirst-degree relatives, Journal of Autism & Developmental Disorders1989; 19(3): 397-407.

[0167] 66. Anderson G.M. Feibel F.C. Wetlaufer L.A. Schlicht K.R. OrtSM. Cohen D.J. Effect of a meal on human whole blood serotonin.Gastroenterology 1985;88 (1Pt 1):86-9.

[0168] While a specific embodiment of the invention has been shown anddescribed in detail to illustrate the application of the principles ofthe invention, it will be understood that the invention may be embodiedotherwise wvithout departing from such principles, and that variousmodifications, alternate constructions, and equivalents will occur tothose skilled in the art given the benefit of this disclosure. Variouschanges and modifications can be made therein without departing from thespirit and scope of the invention. Thus, the invention is not lirrutedto the specific embodiment described herein, but is defined by theappended claims.

What is clainied:
 1. A method for aiding in the diagnosis of autismcomprising the steps of drawing a blood sample from a patient suspectedof suffering from autism, analyzing the level of secretin, andcorrelating said level with norms.
 2. A method for alleviating thesymptoms of autism in an individual exhibiting symptoms of autism,comprising the step of stimulating the secretion of pancreatico-biliaryfluid.
 3. A method as in claim 2 wherein said step of stimulating thesecretion of said pancreatico-biliary fluid stimulates the endogenousrelease of serotonin in said individual.
 4. A method as in claim 2wherein said step of stimulating the secretion of saidpancreatico-biliary fluid comprises the step of administering aneffective amount of secretin to said individual.
 5. A method as in claim4 wherein the amount of secretin administered is between about 2 IU ofsecretin per kg of said individual's body weight.
 6. A method as inclaim 4 wherein the method of administration is selected from the groupconsisting of intravenous, oral, intramuscular, intra-articular,intradermal, subcutaneous; inhalation, and rectal.
 7. A method as inclaim 4 wherein said secretin is chosen from the group consisting ofsynthetic or recombinant human secretin, porcine secretin and bovinesecretin.
 8. A method as in claim 2 wherein said step of stimulatingpancreatico-biliary fluid secretion comprises the step of releasingendogenous secretin in said individual.
 9. A method as in claim 8wherein said step of releasing endogenous secretin comprises the step ofadministering an effective amount of acid to the duodenum.
 10. A methodof claim 8 wherein said step of-releasing endogenous secretin comprisesthe step of administering an effective amount a secretin releasestimulating substance, said substance selected from the group consistingof bile salt; bile acids; 1-phenyl-1-hydroxv-N-pentane (PHP); fats;fatty acids; anti-ulcer pharmaceuticals and herbal extracts.
 11. Amethod of claim 10 wherein said substance is sodium oleate.
 12. A methodof claim 10 wherein said substance is an oleic acid.
 13. A method ofclaim 10 wherein said substance is an anti-ulcer pharmaceutical,
 14. Amethod of claim 13 wherein said anti-ulcer pharmaceutical is selectedfrom the group consisting of PLAUNUTOL™, tetraprenylacetone (TPN),geranyl-geranyl acetone (GGA), and(Z)-2-(4-methylpiperazin-1-yl)-1-[4-(2-phenyl-ethyl)pheny]-eth anoneoxime hydrochloride monohydrate (MCl-727).
 15. A method of claim 10wherein said substance is an herbal extract.
 16. A method of claim 15wherein said herbal extract is licorice root.
 17. A method of claim 10wherein said substance is a bile salt or bile acid. 18 A method of claim10 wherein said substance is I-phenylpentanol orI-phenyl-I-hydroxy-N-pentane (PHP) or a derivative thereof.
 19. A methodof claim 4 wherein said secretin is co-administered with a biologicallyacceptable permeation enhancing agent.
 20. A method of claim 4 whereinsaid secretin is co-administered with a biologically acceptable agentcapable of preventing hydrolysis by colonic bacterial flora or cellularenzymes.